The establishment of polarity within embryonic cells is fundamental to the generation of cell diversity. Because of their relative structural simplicity and distinct polarity markers, early embryos of the nematode Caenorhabditis elegans provide an ideal system for studying cell polarity. It is proposed to study the interplay between cortical and cytoplasmic structures during cell polarization using optical experimental technologies as well as established genetic and molecular techniques. The initial proposed experiments will make use of labeled beads, an actin::GFP construct, and 4D, two-photon fluorescence microscopy to examine cortical flows and actin dynamics in wild-type (polar) and mutant (apolar) embryos. The possible role of calcium signaling in polarity establishment will be examined using, among other techniques, caged version of molecules known to be involved in calcium signaling pathways. These studies will be aimed re-establishing polarity in the apolar mutant by locally uncaging putative signaling molecules. Finally, in order to determine the specific roles of the cytoskeleton in polarity establishment, the cytoskeletal organization will be manipulated by caged versions of cytoskeletal regulators and by the use of optical trapping to specifically localize these regulators with the embryo.